JPH1138324A - Laser scanning microscope - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a laser scanning microscope that can continuously obtain neither too much not too little section images even though the thickness of a sample is not previously calculated. SOLUTION: This microscope is provided with a dichroic mirror 3 reflecting an exciting laser beam and also transmitting fluorescence, a two-dimensional scanning unit 4 two-dimensionally scanning the sample 13 with the exciting laser beam, an objective lens 7 arranged between the two-dimensional scanning unit 4 and the sample 13, a Z-axis controller whose relative position with the sample 13 and the objective lens 7 in an optical axis direction is made movable and a monitor 22 observing the fluorescence. In this case, the relative position is moved in the optical axis direction by a specified distance by driving the Z-axis controller 8, and the image information of the section images acquired by each movement is compared with each other, and the acquisition of the section images is completed based on the compared result of the image information.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a laser scanning microscope.

[0002]

2. Description of the Related Art A confocal laser scanning microscope is composed of a laser light source for irradiating a sample with excitation laser light, a dichroic mirror for separating the excitation laser light and fluorescence emitted from the sample, and a two-dimensional scanning with the excitation laser light. A two-dimensional scan unit, an objective lens disposed between the two-dimensional scan unit and the sample, a condensing lens disposed between the dichroic mirror and the detection device, and a pinhole provided on a focal plane of the condensing lens. A driving device that can move a relative position between the sample and the objective lens in the optical axis direction, and an observation device that observes fluorescence.

An excitation laser beam emitted from a laser light source is reflected by a dichroic mirror, two-dimensionally scanned by a two-dimensional scanning unit, and focused on a sample surface substantially perpendicular to the optical axis by an objective lens.

[0004] The fluorescence generated from the sample by the irradiation of the excitation laser light travels backward along the optical path from the objective lens to the scanning mirror together with the excitation laser light, and is separated from the excitation laser light by the dichroic mirror.

[0005] The fluorescence transmitted through the dichroic mirror is
The light is focused on a pinhole arranged in the optical path by the focusing lens. At this time, only the fluorescence emitted from the imaging portion of the sample passes through the pinhole, is received by the detection device, and is converted into an electric signal corresponding to the light amount by the detection device.

[0006] A very thin slice image of the sample can be obtained by subjecting the electric signal and the scanning position information to predetermined processing using a computer. At this time, by driving the driving device to move the focal plane in the optical axis direction to change the distance (focus) between the objective lens and the sample, it is possible to continuously acquire the slice images on each focal plane.

In order to obtain a slice image of the entire sample, the thickness of the sample is determined in advance, and conditions such as the focal plane movement interval and the number of slice images to be obtained are stored in a memory in advance in accordance with the thickness. Need to be kept.

[0008]

However, in order to obtain the thickness of the sample, the position of the stage in the Z direction (optical axis direction) at the time of acquiring the first image (the uppermost portion of the sample) and the last position (the direction of the sample) are obtained. The thickness of the sample needs to be calculated in advance from the position of the stage in the Z direction at the time of image acquisition (at the bottom), which is troublesome.

If the thickness of the sample is not accurately determined,
The number of continuously acquired slice images may be too small or too large.

FIGS. 4 and 5 are diagrams for explaining the relationship between the thickness of the sample and the number of slice images.

For example, when the thickness of the sample obtained by the calculation is smaller than the actual sample 113, the required number of slice images 113A in the thickness direction of the sample 113 cannot be obtained, and the sample 113 cannot be obtained. Image information 113B is insufficient (see FIG. 4).

Conversely, if the thickness of the sample obtained by calculation is larger than the actual thickness of the sample 113, a meaningless slice image 113A is acquired as image information 113B of the sample 113 (see FIG. 5).

Further, in order to determine the thickness of the sample 113, the same portion of the sample 113 is repeatedly scanned by laser light. Therefore, in the case of a sample which emits fluorescence by irradiation with laser light, damage to the sample or fading of the fluorescence There is a problem that becomes large.

The present invention has been made in view of such circumstances, and an object of the present invention is to provide a laser scanning microscope capable of continuously acquiring a slice image of the entire sample without excess or deficiency without previously calculating the thickness of the sample. To provide.

[0015]

According to a first aspect of the present invention, there is provided a laser scanning microscope which reflects an excitation laser beam and transmits fluorescence.
Scanning means for two-dimensionally scanning the excitation laser light on the sample, an objective lens arranged between the scanning means and the sample, and a movable relative position in the optical axis direction between the sample and the objective lens Laser scanning microscope, comprising: a driving unit that receives the fluorescence transmitted through the light separating unit and an observation unit that obtains a slice image of the sample. The driving unit drives the driving unit to move the relative position in the optical axis direction. Control means for moving the image by a predetermined distance, and for each movement, comparing the image information of the slice images acquired before and after the movement, and ending the acquisition of the slice image based on the comparison result of the image information. It is characterized by having.

The driving means is driven to move the relative position by a predetermined distance in the direction of the optical axis, and the image information of the slice images acquired at each movement is compared. Based on the comparison result of the image information, the slice information is obtained. Since the acquisition of the image is terminated, while the sample is present and the image information of the previously acquired section image and the image information of the later acquired section image are different, the movement of the focal plane and the acquisition of the section image are performed, When the sample does not exist and the image information of the adjacent slice images becomes the same, the movement in the optical axis direction and the acquisition of the slice images are completed.

According to a second aspect of the present invention, in the laser scanning microscope according to the first aspect, the image information is luminance information of each pixel, and the luminance information before the comparison is predetermined. When the value having the value equal to or more than the threshold value is equal to or more than the predetermined ratio, the control unit continues acquiring the slice image.

If the luminance information before comparison has a value equal to or greater than a predetermined threshold value and equal to or greater than a predetermined ratio, it is determined that a sample is present, and the slice image acquisition is continued.

A laser scanning microscope according to a third aspect of the present invention is the laser scanning microscope according to the first aspect, wherein the image information is luminance information of each pixel, and the comparison result is a value equal to or less than a predetermined threshold. When it has the predetermined ratio or more, the control means ends the acquisition of the slice image.

When the comparison result has a value equal to or less than a predetermined threshold value or more than a predetermined ratio, it is determined that the sample does not exist, and the acquisition of the slice image ends.

[0021]

Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a block diagram of a confocal laser scanning microscope according to a first embodiment of the present invention.

The confocal laser scanning microscope uses a laser light source 1
, A beam expander 2, a dichroic mirror (light separating means) 3, a two-dimensional scanning unit (scanning means) 4, a scanner controller 5, and a condenser lens 6.
, Objective lens 7, Z-axis controller (driving means) 8
, An absorption filter 9, a condenser lens 10, a pinhole filter 11, a photodetector 12, a computer (control means) 20, an image processing circuit 21, and a monitor (observation means) 22.

The light source 1 emits an excitation laser beam (hereinafter, referred to as a laser beam) and irradiates a sample 13 mounted on a stage (not shown).

The beam expander 2 expands the laser light to a size that satisfies the pupil plane of the objective lens.

The dichroic mirror 2 does not transmit the laser beam but transmits the fluorescence emitted from the sample 13.

The two-dimensional scanning unit 4 includes a horizontal scanner 4A and a vertical scanner 4B that perform scanning in the X and Y directions by the scanner controller 5.

The scanner controller 5 is a computer 2
0 and the vertical synchronization signal 2a and the vertical synchronization signal 2
1b, a horizontal scanner 4A and a vertical scanner 4B.
And control.

The objective lens 7 is a two-dimensional scanning unit 4
And the sample 13, and is moved in the optical axis direction with respect to the sample 13 by the Z-axis controller 8.

The Z-axis controller 8 is controlled by a movement signal 20b output from the computer 20 and instructing a movement interval in the optical axis direction and a movement direction a.

As the Z-axis controller 8, for example, DC
A servo motor can be used.

The condenser lens 10 is a dichroic mirror 3
The light that has been separated and passed through the absorption filter 9 is collected.

The pinhole filter 11 is provided at a position conjugate with the focal plane of the objective lens 7 and allows only light emitted from the image forming portion of the sample 13 to pass.

The fluorescence detector 12 is a pinhole filter 11
The light of the sample 13 that has passed through is detected, and the input light is converted into a signal 12a representing the light intensity.

The image processing circuit 21 includes an A / D converter, a frame memory, a D / A converter, and other circuits for imaging.

The image processing circuit 21 records a section image of the sample in a frame memory based on the signal 12a of the photodetector 12 and the pixel clock signal 20a output from the computer 20, and monitors the acquired section image. 22. Further, the image information of the acquired slice image is transmitted to the computer 20.

FIG. 2 is a flowchart for explaining a method of acquiring a section image by the computer 20. Note that FIG.
In S1, S1 to S8 indicate each step.

First, the user inputs information such as the movement interval in the optical axis direction from a keyboard (not shown). This information is stored in the memory 20A.

The operator drives the Z-axis controller 8,
The operator focuses on the image at the upper end of the sample 13 while watching the image on the monitor 22, and sets the position in the Z direction (optical axis direction) as the first slice image. Two-dimensional scanning is performed by the two-dimensional scanning unit 4 at this focal position, and the image information of the slice image is stored in the memory 20A. (S1).

Next, the computer 20 drives the Z-axis controller 8 to lower the objective lens 7 by a predetermined distance in the optical axis direction (S2).

At this new focal position, the two-dimensional scanning of the sample 13 is performed by the two-dimensional scanning unit 4 to obtain image information of the slice image (S3).

The computer 20 obtains the pixel value of each pixel from the image information obtained from the image processing circuit 21 and determines the number of pixels at which the pixel value (luminance value is quantified in the range of 0 to 255) is 20 or more. It is determined whether or not the ratio occupied by is less than 10% of the total number of pixels (S4).

If it is determined in S4 that the ratio of the number of pixels whose pixel value is 20 or more is not less than 10% of the total number of pixels, the image information of the slice image acquired this time is stored in the memory 20A (S5). ).

If it is determined in S4 that the ratio of the number of pixels having a pixel value of 20 or more is 10% or less of the total number of pixels, the image information of the previously obtained section image is obtained from the image information of the currently obtained section image. The image information is subtracted (subtraction of the pixel value of the corresponding pixel) (S6).

Next, the computer 20 obtains the number of pixels in which the absolute value of the pixel value subtracted in S6 becomes 10 or less, and the ratio of the number of pixels is 90% of the total number of pixels.
% Is determined (S7).

If the ratio of the number of pixels whose absolute value of the pixel value is 10 or less is not 90% or more of the total number of pixels, the obtained image information of the slice image is stored in the memory 20A (S8).

If the ratio of the number of pixels whose absolute value of the pixel value is 10 or less is 90% or more of the total number of pixels, it is determined that there is no sample 13 and the acquisition of the image information of the slice image is terminated. (S9).

According to the first embodiment, the following effects can be obtained.

While the sample 13 is present and the image information between the slice images is different, movement of the focal plane and acquisition of the slice image are performed. When they become the same, the movement in the Z direction and the acquisition of the slice image are terminated, so that the image information of the slice image can be acquired without excess or deficiency without previously calculating the thickness of the sample 13 in advance.

It is not necessary to repeatedly scan the same portion of the sample 13 to calculate the thickness of the sample 13 in advance, and only one scan is required. Therefore, even in the case of the sample 13 which emits fluorescence, damage and fading of the fluorescence can be prevented. Can be suppressed.

If the ratio of the number of pixels whose pixel value is 20 or more is not less than 10% of the total number of pixels, the image information of the intercept image is obtained, so that the pixel value difference between the intercept images is small. However, there is no inconvenience that the acquisition of the slice image is terminated and the image information is insufficient.

If the ratio of the number of pixels having a pixel value of 10 or less is 90% or more of the total number of pixels, the acquisition of the image information of the slice image is terminated, and the image information of the slice image contains many noise components. Thus, even when the pixel value difference between the slice images does not become zero, the acquisition of the slice images is surely terminated, and the meaningless image information is not obtained.

FIG. 3 is a flowchart for explaining another method of acquiring a section image by the computer 20 (second embodiment). In FIG. 3, S11 to S18 indicate each step.

First, the user inputs information such as the movement interval in the optical axis direction from a keyboard (not shown). This information is stored in the memory 20A.

The operator drives the Z-axis controller 8, and
The operator focuses on the image at the upper end of the sample 13 while watching the image on the monitor 22, and sets the position in the Z direction (optical axis direction) as the first slice image. Two-dimensional scanning is performed by the two-dimensional scanning unit 4 at this focal position, and the image information of the slice image is stored in the memory 20A. (S11).

Next, the computer 20 drives the Z-axis controller 8 to lower the objective lens 7 by a predetermined distance in the optical axis direction (S12).

At this new focus position, the two-dimensional scanning of the sample 13 is performed by the two-dimensional scanning unit 4 to obtain image information of a slice image (S13).

The computer 20 obtains the pixel value of each pixel from the image information obtained from the image processing circuit 21,
It is determined whether or not the proportion of the number of pixels whose pixel value is 20 or more is 10% or less of the total number of pixels (S
14).

If it is determined in S14 that the ratio of the number of pixels having a pixel value of 20 or more is not less than 10% of the total number of pixels, the image information of the slice image acquired this time is stored in the memory 20A (S15). ).

If it is determined in S14 that the ratio of the number of pixels having a pixel value of 20 or more is 10% or less of the total number of pixels, the image information of the slice image acquired this time and the image information of the slice image acquired last time are determined. Division with the image information (division of the pixel value of the corresponding pixel) is performed (S16).

Next, the computer 20 obtains the number of pixels for which the result of the division in S16 is 1 ± 0.2, and determines whether or not the ratio of the number of pixels is 90% or more of the total number of pixels (step S16). S17).

If the ratio of the number of pixels whose division result is 1 ± 0.2 is not 90% or more of the total number of pixels, the obtained image information of the slice image is stored in the memory 20A (S18).

If the ratio of the number of pixels whose division result is 1 ± 0.2 is 90% or more of the total number of pixels, it is determined that there is no sample 13 and the acquisition of the image information of the slice image is terminated. (S19).

According to the second embodiment, the same effects as in the first embodiment can be exhibited.

Although the objective lens is used in each of the above embodiments, a piezo element, a stepping motor, an ultrasonic motor, or the like may be used instead of a DC motor.

[0066]

As described above, according to the laser scanning microscope of the first aspect of the present invention, the sample exists, and the image information of the previously acquired section image differs from that of the later acquired section image. While moving, move the focal plane and acquire the section image,
When the sample disappears and the image information of the previously acquired section image and the image information of the section image acquired later become the same, the movement in the optical axis direction and the acquisition of the section image are terminated. It is possible to acquire the image information of the slice image without excess or deficiency without performing the calculation.

Even in the case of a sample that emits fluorescence, the scanning of the sample only needs to be performed once, so that damage to the sample and fading of the fluorescence can be suppressed.

According to the laser scanning microscope of the second aspect of the invention, when the luminance information before comparison has a value equal to or more than a predetermined threshold value and a predetermined ratio or more, it is determined that a sample is present, and the slice image is determined. Since the acquisition is continued, even if the difference between the pixel values between the slice images is small, the acquisition of the slice image is terminated, and there is no inconvenience that the image information is insufficient.

According to the laser scanning microscope of the third aspect of the present invention, when the comparison result has a value equal to or less than a predetermined threshold value and a predetermined ratio or more, it is determined that the sample does not exist,
Since the acquisition of the slice image is terminated, even if the image information of the slice image contains a lot of noise components and the pixel value difference between the slice images does not become zero, the acquisition of the slice image is surely terminated and is meaningless. There is no inconvenience of acquiring image information.

[Brief description of the drawings]

FIG. 1 is a block diagram of a confocal laser scanning microscope according to a first embodiment of the present invention.

FIG. 2 is a flowchart illustrating a method of acquiring a section image by a computer.

FIG. 3 is a flowchart illustrating another method for acquiring a slice image by a computer.

FIG. 4 is a diagram for explaining the relationship between the thickness of a sample and the number of slice images.

FIG. 5 is a diagram illustrating the relationship between the thickness of a sample and the number of slice images.

[Explanation of symbols]

 Reference Signs List 1 laser light source 2 beam expander 3 dichroic mirror (light separating means) 4 two-dimensional scan unit (scanning means) 7 objective lens 8 Z-axis controller (driving means) 11 pinhole filter 20 computer (control means) 22 monitor (observing means) )

Claims (3)

[Claims] 1. A light separating unit that reflects an excitation laser beam and transmits fluorescence, a scanning unit that two-dimensionally scans the excitation laser beam on a sample, and is disposed between the scanning unit and the sample. An objective lens, a driving unit capable of moving a relative position of the sample and the objective lens in an optical axis direction, and an observation unit that receives fluorescence transmitted through the light separating unit and acquires a slice image of the sample. In the provided laser scanning microscope, by driving the driving means to move the relative position by a predetermined distance in the optical axis direction, for each movement, comparing the image information between the slice images obtained before and after the movement, A laser scanning microscope comprising a control unit for terminating the acquisition of the slice image based on the comparison result of the image information. 2. The method according to claim 1, wherein the image information is luminance information of each pixel, and when the luminance information before the comparison has a value equal to or more than a predetermined threshold value and a predetermined ratio or more, the control unit obtains a slice image. 2. The laser scanning microscope according to claim 1, wherein: 3. The method according to claim 2, wherein the image information is luminance information of each pixel, and when the comparison result has a value equal to or less than a predetermined threshold value and equal to or more than a predetermined ratio, the control unit ends the acquisition of the intercept image. The laser scanning microscope according to claim 1, wherein